Analytical characterization and purification of a commercial extract of enzymes: A case study

•CALB was purified from additives and impurities present in a commercial extract.•Protein quantification was achieved by means of a standard curve of pure CALB.•A rational and reliable strategy for CALB purification involves two chromatographic steps.•The process was performed using a volatile buffer, easily removed by lyophilization.

This paper presents a rational strategy to identify and quantify the components of a commercial extract of the lipase B of Candida antarctica that can be extended to the analytical investigation of other crude extracts of enzymes. These information provided the fundamental knowledge for the development of a methodology to obtain highly pure and catalytically active CALB enzyme.The commercial extract Lipozyme® was subjected to a series of analytical techniques that allowed determining the presence of a non-soluble fraction; nucleic acids; benzoate and sorbate species and a mixture of three proteins. Particularly, it is worth noticing that the Bradford assay using CALB as standard instead of BSA proved to be a more reliable and accurate methodology to quantify the protein content of the assayed enzymatic samples. Size exclusion chromatography coupled with anionic exchange chromatography using a non-conventional, easy to remove buffer system such as ammonia–ammonium acetate afforded a sample that retains 47% of the proteins (being CALB the only enzymatic component of the purified sample) with a hydrolytic activity higher than the crude extract.

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